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Ayaka Kadotani, [Gen Hayase](https://orcid.org/0000-0003-1970-6129), Daisuke Yoshino

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[Geometrically engineered organoid units and their assembly for pre-construction of organ structures](https://mdr.nims.go.jp/datasets/bbc54f14-cf36-4104-a74e-0b360f690523)

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Geometrically engineered organoid units and their assembly for pre-construction of organ structuresViewOnlineExportCitationRESEARCH ARTICLE |  NOVEMBER 26 2024Geometrically engineered organoid units and their assemblyfor pre-construction of organ structures Ayaka Kadotani  ; Gen Hayase   ; Daisuke Yoshino  APL Bioeng. 8, 046112 (2024)https://doi.org/10.1063/5.0222866Articles You May Be Interested InOctoShaker: A versatile robotic biomechanical agitator for cellular and organoid researchRev. Sci. Instrum. (December 2023)The role of nonlinear mechanical properties of biomimetic hydrogels for organoid growthBiophysics Rev. (June 2021)Revolutionizing biomedical research: The imperative need for heart–kidney-connected organoidsAPL Bioeng. (February 2024)  02 December 2024 11:46:41https://pubs.aip.org/aip/apb/article/8/4/046112/3322496/Geometrically-engineered-organoid-units-and-theirhttps://pubs.aip.org/aip/apb/article/8/4/046112/3322496/Geometrically-engineered-organoid-units-and-their?pdfCoverIconEvent=citejavascript:;https://orcid.org/0009-0007-3285-5280javascript:;https://orcid.org/0000-0003-1970-6129javascript:;https://orcid.org/0000-0002-2948-1210https://crossmark.crossref.org/dialog/?doi=10.1063/5.0222866&domain=pdf&date_stamp=2024-11-26https://doi.org/10.1063/5.0222866https://pubs.aip.org/aip/rsi/article/94/12/124104/2930525/OctoShaker-A-versatile-robotic-biomechanicalhttps://pubs.aip.org/aip/bpr/article/2/2/021401/150254/The-role-of-nonlinear-mechanical-properties-ofhttps://pubs.aip.org/aip/apb/article/8/1/010902/3267505/Revolutionizing-biomedical-research-The-imperativehttps://e-11492.adzerk.net/r?e=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&s=Zi5Cl4fRHgnItOR2DZCh4aTN83sGeometrically engineered organoid unitsand their assembly for pre-constructionof organ structuresCite as: APL Bioeng. 8, 046112 (2024); doi: 10.1063/5.0222866Submitted: 11 June 2024 . Accepted: 11 November 2024 .Published Online: 26 November 2024Ayaka Kadotani,1 Gen Hayase,2,,a) and Daisuke Yoshino1,3,,a)AFFILIATIONS1Department of Biomedical Engineering, Graduate School of Engineering, Tokyo University of Agriculture and Technology,2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan2Research Center for Electronic and Optical Materials, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044,Japan3Division of Advanced Applied Physics, Institute of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho,Koganei, Tokyo 184-8588, Japana)Authors to whom correspondence should be addressed: gen@aerogel.jp and dyoshino@go.tuat.ac.jpABSTRACTRegenerative medicine is moving from the nascent to the transitional stage as researchers are actively engaged in creating mini-organs frompluripotent stem cells to construct artificial models of physiological and pathological conditions. Currently, mini-organs can express higher-order functions, but their size is limited to the order of a few millimeters. Therefore, one of the ultimate goals of regenerative medicine,“organ replication and transplantation with organoid,” remains a major obstacle. Three-dimensional (3D) bioprinting technology is expectedto be an innovative breakthrough in this field, but various issues have been raised, such as cell damage, versatility of bioink, and printingtime. In this study, we established a method for fabricating, connecting, and assembling organoid units of various shapes independent of celltype, extracellular matrix, and adhesive composition (unit construction method). We also fabricated kidney tissue-like structures using threetypes of parenchymal and interstitial cells that compose the human kidney and obtained findings suggesting the possibility of crosstalkbetween the units. This study mainly focuses on methods for reproducing the structure of organs, and there are still issues to be addressed interms of the expression of their higher-order functions. We anticipate that engineering innovation based on this technique will bring us closerto the realization of highly efficient and rapid fabrication of full-scale organoids that can withstand organ transplantation.VC 2024 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International (CC BY-NC-ND) license (https://creativecommons.org/licenses/by-nc-nd/4.0/). https://doi.org/10.1063/5.0222866INTRODUCTIONThe development of three-dimensional (3D) culture systems,organoids, has been the most exciting advance in medical and biologi-cal fields in the last two decades since the invention of iPS cells.1–6 Thisorganoid system has enabled the modeling of genetic, degenerative,and cancer diseases that were difficult to reproduce in vitro7–11 and hasbeen expected to lead to innovative advances in establishing medicaltreatments. Organoids can express the higher-order functions oforgans,12 but are currently limited to a few millimeters in size, referredto as “mini-organs” are called. Therefore, one of the ultimate goals ofregenerative medicine, “organ replication and transplantation usingorganoids,” faces a major obstacle.Currently, 3D bioprinting is the most promising method for pro-ducing artificial tissue that can be used as organs for transplantation.Extrusion-based bioprinting (EBB), in which a solution containing liv-ing cells, bioink, is extruded and layered in a 3D space such as a 3Dprinter, is a mainstream method.13 EBB has the advantage of beingable to form complex structures, including internal architecture, with-out the need to prepare molds with cell-dense solutions as bioinks.14Conversely, problems with EBB include cellular damage due to pres-sure and shear forces during bioink ejection, the difficulty of develop-ing bioinks with appropriate mechanical, structural, and biologicalproperties, and the long time required to print full-size organs.15–17Volumetric bioprinting (VBP), unlike conventional methods (i.e.,APL Bioeng. 8, 046112 (2024); doi: 10.1063/5.0222866 8, 046112-1VC Author(s) 2024APL Bioengineering ARTICLE pubs.aip.org/aip/apb 02 December 2024 11:46:41https://doi.org/10.1063/5.0222866https://doi.org/10.1063/5.0222866https://doi.org/10.1063/5.0222866https://www.pubs.aip.org/action/showCitFormats?type=show&doi=10.1063/5.0222866http://crossmark.crossref.org/dialog/?doi=10.1063/5.0222866&domain=pdf&date_stamp=2024-11-26https://orcid.org/0009-0007-3285-5280https://orcid.org/0000-0003-1970-6129https://orcid.org/0000-0002-2948-1210mailto:gen@aerogel.jpmailto:dyoshino@go.tuat.ac.jphttps://creativecommons.org/licenses/by-nc-nd/4.0/https://doi.org/10.1063/5.0222866pubs.aip.org/aip/apblayer-by-layer stacking), creates an object by photo cross linking theresin by exposing it to a calculated two-dimensional (2D) light patternwhile rotating a transparent container.18 As a result, it takes only a fewseconds to a few minutes to fabricate a model, regardless of size. Thefeatures of short fabrication time and nozzle-less fabrication do notaffect cell viability and functionality and provide high resolution (up totens of micrometers).19 Hence, VBP is a new and promising techniquethat overcomes several of EBB’s limitations. However, it is difficult toprecisely control the position and density distribution of cells in thepre-cured gel with current methods,19,20 and it is expected to be chal-lenging to construct full-size organs composed of different cells andextracellular substrates. Therefore, it is desirable to develop a techniquefor artificially constructing organ-size 3D tissues with high efficiencyand yield.To address these current problems, a method of 3D tissue engi-neering using organoid building blocks (OBBs), i.e., stacking OBBssuch as spheroids and organoids to reproduce organ-specific functions,has been developed.21 The stacking of organoids and spheroids rang-ing in size from several hundred lm3 to 1mm3 is expected to signifi-cantly reduce the printing time currently required for the spatialarrangement of single cells, bringing us closer to the rapid constructionof human tissues. In particular, the OBB has recently been introducedinto a 3D bioprinter, enabling large-scale organoid-like structures,assembloid, by realizing rapid spatial arrangement.21–24 Although thistechnology solves the heterogeneity of iPS cell-derived organoids,more is needed to address the limitations of the types of bioinks, thesize of the OBBs that can be used, the complicated spatial arrangementdue to the basic shape of the block, such as a sphere, and the changesin bioink properties associated with printing due to the use of a 3Dbioprinter.In contrast, we conceptualize the establishment of a method toassemble organoid units of various shapes that are pre-divided intobasic organ elements like construction toys (unit constructionmethod). The unit construction method has several advantages overconventional technologies. First, there are no limitations on the typesof extracellular matrix (ECM) that can be used, and it is cell-friendlyby avoiding damage from pressure, etc., because it does not involveextrusion molding like a 3D printer. Second, the method allows us toconstruct organoids containing multiple cells and ECMs with high effi-ciency by fabricating each unit from different cells and ECMs and thenassembling them. Furthermore, we can easily reproduce complexstructures by combining units that are not limited to simple geome-tries, such as spheres and sheets. This method leads to a highly efficientand rapid technique for producing full-scale organoids that can surviveorgan transplantation. In the future, we envision the production oforganoids of transplantable size. In particular, we are focusing on theconstruction of full-size kidney organoids as one of the treatmentoptions for kidney diseases that are difficult to cure with drugs. Here,we show our method for fabricating organoid units with six differentshapes and the cellular dynamics inside the units when stacked, as wellas the results of forming and assembling the units using three types ofparenchymal and interstitial cells that compose the human kidney.RESULTS AND DISCUSSIONFabrication of organoid units with various geometriesWe first selected and fabricated the organoid units with thegeometry necessary to reproduce the nephrons and other structures inthe human kidney. We confirmed that we had no problems releasingthe organoid units with various shapes from the molds, and could fab-ricate them with constant reproducibility [Figs. 1(a) and 1(b), supple-mentary Fig. 1]. Organoid units made with MDA-MB-231 cellscontracted in size as they matured [Fig. 1(c)]. Previous studies havealso reported this phenomenon, which is partly due to actomyosincontractility,25 typical of highly invasive or motile cell types. Notably,even organoids with complex shapes contracted while maintainingtheir overall shape [Figs. 1(d)–1(i)]. The bead ring-shaped units alsoshowed their contraction without rupture of their respective connect-ing parts.Cell proliferation inside and on the surface of the organoids wasobserved as the culture duration progressed [Figs. 2(a)–2(f)]. As theorganoid units, which are thicker than the sheet types [Fig. 2(d)],matured, the cells inside them became denser and underwent celldeath. This is caused by the dense cellular area on the organoid surfacelayer, which acts like a shell and blocks the oxygen and nutrient supplyto the inner cells.26,27 This notion is supported by the results of an eval-uation of cell death within the 2mm-diameter spherical organoid unitsstained with Hoechst, Annexin V (a marker of apoptosis), andEthidium homodimer III (a marker of necrosis). The percentage ofEthidium homodimer III-positive cells increased with the number ofdays in culture for both types of organoid units [MDA-MB-231, Figs.2(g) and 2(h); HRGEpCs, Figs. 2(i) and 2(j)]. In contrast, the percent-age of Annexin V-positive cells did not change with culture duration[Fig. 2(j)]. Cell death within organoids is a common problem in previ-ous organoid studies. It can be circumvented by properly arrangingsupply channels such as the vascular networks.28,29 This study allowsus to easily fabricate organoid units that reproduce the vascular system,as described below. By placing these units inside during assembly, alarge-scale organoid that can be cultured for a long period of time isexpected to be constructed.We then assembled the various organoid units to evaluate a unitconstruction method. The substrate, collagen solution, was applied tothe area with a micropipette and incubated for 2–5min to allow theunits to bond and stack [Fig. 3(a)]. Once the four cylindrical unitswere joined together to form a structure, we were able to grab one endof the structure with tweezers and pull it up, holding the structure inplace without breaking it apart [Figs. 3(b) and 3(c), supplementaryMovie 1]. Even rectangular units could be easily glued together[Fig. 3(d)], and more units could be stacked on top of the glued units[Fig. 3(e)]. The composite of the stacked units did not collapse whenshaken (supplementary Movies 2 and 3). After assembly, continuedculture is expected to induce proliferation and organization of cellswithin the units, leading to crosstalk between the units [Fig. 3(f)].However, there are still some problems with the bonding. In this study,the ECM solution that acts as an adhesive is applied by a micropipette,which results in a large loss due to the inability to apply the solution ina spot manner. This causes a large displacement in the stacking of mul-tiple units. Thus, it suggests that the realization of micro-spot bondingand precise stacking (e.g., micromanipulation system) is necessary forthe future construction of organ structures.Construction and assembly of kidney glomerulartissue-like organoid unitsThe use of easy-to-handle cancer cell lines is suitable for validat-ing the production of living parts of various shapes for full-scaleAPL Bioengineering ARTICLE pubs.aip.org/aip/apbAPL Bioeng. 8, 046112 (2024); doi: 10.1063/5.0222866 8, 046112-2VC Author(s) 2024 02 December 2024 11:46:41pubs.aip.org/aip/apbFIG. 1. Fabrication of organoid units with various geometries. (a) MDA-MB-231 organoid units immediately after fabrication with typical shapes (n¼ 5). Scale bars, 1 mm. (b)Table of shape reproducibility in organoid fabrication immediately after they were fabricated (day 0) (mean 6 SD, n¼ 5). (c) Time-sequenced brightfield images of MDA-MB-231 organoid units of various geometries (n¼ 3). Scale bars, 1 mm. (d)–(i) Dimensional changes of MDA-MB-231 organoid units of various shapes and their ratios with cultureduration [(d) bead ring; (e) cylinder; (f) rectangular solid; (g) sheet; (h) sphere; and (i) spherical shell, mean6 SD, n¼ 3]. For the spherical shells, we evaluated the case wherea layer of cells was placed on the outside and inside, respectively.APL Bioengineering ARTICLE pubs.aip.org/aip/apbAPL Bioeng. 8, 046112 (2024); doi: 10.1063/5.0222866 8, 046112-3VC Author(s) 2024 02 December 2024 11:46:41pubs.aip.org/aip/apborganoid construction. However, it is a considerable leap in researchand development steps to construct something that functions as anorgan (especially a kidney). Here, we examined the validity of our pro-posed method (unit construction) by constructing and assemblingunits with organ-like structures under a tri-culture of parenchymaland interstitial cells (i.e., renal glomerular epithelial cells, mesangialcells, and vascular endothelial cells) that consist of the kidney glomeru-lus. Tri-cultured organoid units exhibited a tissue-like structure, whereHRGEpCs were complexly intertwined around HUVECs, whichformed a vascular network, and NHMCs were interspersed aroundthem [Fig. 4(a)]. These findings are supported by the co-localization ofmarker proteins of each cell type [i.e., Pearson’s correlation coefficient;Fig. 4(b)]. Integrating these two sets of data [Figs. 4(a) and 4(b)] pro-vides a rough outline of the internal structure in the fabricatedunit. Compared to the histological structure of a normal humankidney,30–32 the morphology and distribution of NHMCs andHUVECs in the fabricated organoid unit were similar to those ofmesangial cells and endothelial cells in the renal glomerulus. However,HRGEpCs, which become podocytes in a normal kidney, were in closespatial proximity to HUVECs but did not form a membrane-like struc-ture around them. The reason for this may be that the tissue did notmature well under the culture conditions in this study due to the lackof orientation of the units. We should further investigate the challengesin structural mimicry (i.e., the formation of membrane-like structuresby HRGEpCs) while analyzing the spatial relationships of cells in moredetail in the future.The vascular network formed by the HUVECs decreased with theprogression of the culture, and by day 5, the internal network hadalmost disappeared [Fig. 4(c)]. This is most likely due to the fact thatthe network was recognized as unnecessary because the units were cul-tured statically. This notion is supported by reports that turnover ofthe vascular network is induced when the culture medium is not per-fused.33,34 After the tri-cultured organoid units were bonded to eachother with a substrate solution containing interstitial cells (HUVECs),the vascular network formed by the HUVECs seemed to connect thetwo units [Fig. 4(d)]. Crosstalk between units will be possible if thestructures with stacked units are cultured under appropriate condi-tions, including perfusion of the medium into the vascular network,and we can expect the stacked structures to mature as tissues.However, the vascular network did not function properly and contin-ued to mature, and it began to disappear after the third day. In orderto maintain long-term culture while inducing crosstalk between cellsin multiple units, a hierarchical vascular network that functions as asupply channel for oxygen and nutrients is essential. An open questionfor the future is how to maintain the vascular network formed withinthe organoid complex and to mature it into a hierarchical structure.Assembly of organoid units with different geometries,aiming to construct a complex structureTo reproduce the complex structure of human organs, we need tocombine organoid units with different shapes. Finally, we investigatedthe scalability of the proposed method by assembling bead-ring-shaped and cylinder-shaped units. Using tweezers, we gently lifted thebead ring-shaped unit and inserted the cylinder-shaped unit into itscentral space [Fig. 5(a)]. The contact points between the units werebonded with collagen solution or protein solution (mixture of collagenand Matrigel), and the complexes were cultured for 5 days. For thepractice, we started by assembling organoid units prepared withMDA-MB-231 cells, which are easy to handle [Figs. 5(b) and 5(c)].During the 5-day culture, we observed that the units did not peel offfrom each other, but gradually contracted while maintaining theirshape. As with other results, the cells inside the organoid units gradu-ally underwent cell death as the culture duration progressed. Althoughit is difficult to see in the bright field observation results [Fig. 5(b)], thefluorescence imaging results [Fig. 5(c)] show that the spherical beadswere arranged in a 3D manner in front of and behind the cylinder-shaped units. We were also able to construct a 3D structure in thesame way using organoid units mimicking kidney glomerular tissue(i.e., organoids tri-cultured with NHMCs, HUVECs, and HRGEpCs)[Fig. 5(d)]. Summarizing the above-mentioned results, we can con-clude that the proposed method has the potential to replicate complexhuman organ structures by assembling organoid basic units.Our proposed unit construction method can construct tissue-likestructures by fabricating and assembling organoid units of variousshapes and sizes. The unit construction method is completely differentfrom 3D bioprinting (i.e., it does not require bioink) and has no limita-tions on the types of ECMs that can be used. We can fabricate unitsusing a similar protocol even in an environment with different cellsand ECMs, and we can construct organoids containing multiple cellsand ECMs with high efficiency by simply assembling them. Of course,a detailed micromanipulation system is required for their assembly,but this is suitable for constructing organoids that mimic real organs,which are complex and large structures, because it is also possible tocombine units with different geometries. Cell viability can also bemaintained at a high level by adequately arranging the vascular net-work that supplies oxygen and nutrients inside under appropriate con-ditions. Our approach is similar in some respects to the developmentalprocess of tissues and organs because we subdivide the structure of thetarget organ into elements (units), utilize self-organization of cell clus-ters at the unit level, and finally assemble them. In contrast, conven-tional bioprinting technologies21,35,36 have the advantage of being ableto arrange cell clusters in space, but still need to solve the issues of bio-ink development and its influence on cell viability and function, as wellas the time required to construct full-scale organoids. In addition, incases where multiple cell types are used in bioprinting, it is easy toforesee various obstacles to optimizing printing conditions. In general,however, we believe that the greatest challenge for both conventionalbioprinting and our method is the expression of higher-order func-tions in the target organs37–39 in order to realize the construction oftransplantable organoids. Furthermore, we may have to mimic themechanical properties of tissues, especially the interstitium, in thefuture, considering that cells sense the surrounding mechanical field,including the extracellular matrix, and change their functions.40–43CONCLUSIONSIn conclusion, we have proposed a method for fabricating andassembling organoid units of various shapes with a size of a few mm asbasic elements for the realization of full-scale implantable organoids inthe future (Fig. 6). Our method is not limited by the ECM types andorganoid unit size because it does not use a 3D bioprinter. Therefore,the users can choose the ECM suitable for the organoid unit to be con-structed. Moreover, there is no restriction on the adhesive used in ourmethod, so by selecting the appropriate adhesive, it is possible to fabri-cate large constructions such as assembloids at high speed, which isexpected to achieve the “highly efficient and high-volume 3D tissueAPL Bioengineering ARTICLE pubs.aip.org/aip/apbAPL Bioeng. 8, 046112 (2024); doi: 10.1063/5.0222866 8, 046112-4VC Author(s) 2024 02 December 2024 11:46:41pubs.aip.org/aip/apbFIG. 2. Cell dynamics inside the fabricated organoid units. (a)–(f) Live fluorescence images of MDA-MB-231 organoid units, expressing GFP in the cytoplasm, with variousgeometries [(a) bead ring; (b) cylinder; (c) rectangular solid; (d) sheet; (e) sphere; and (f) spherical shell, mean 6 SD, n¼ 3]. Upper row: wide-field fluorescence images(raw data), and lower row: images processed by the THUNDER imaging system with instant computational clearing (ICC) and extended depth of field (EDF). Dashed linesindicate the outline of the outer shell formed by the collagen gel (f). Scale bars, 1 mm. (g) Representative results of live/dead assays for MDA-MB-231 organoid units(n¼ 5). The MDA-MB-231 cells express GFP in their cytoplasm, and we therefore performed necrosis detection using Ethidium homodimer III (EthD-III). Scale bars,500 lm. (h) Quantification of EthD-III-positive cells in the organoid units (mean 6 SD, n¼ 5). The quantified results indicate the percentage of EthD-III-positive cells percell number (EthD-III/Hoechst ratio). (i) Representative results of live/dead assays for organoid units prepared with HRGEpCs (n¼ 3). Scale bars, 500 lm. (j)Quantification of EthD-III-positive and Annexin-V positive cells in the organoid units (mean 6 SD, n¼ 5). The quantified results indicate the percentage of EthD-III-positiveand Annexin V-positive cells per cell number (EthD-III/Hoechst ratio and Annexin V/Hoechst ratio).APL Bioengineering ARTICLE pubs.aip.org/aip/apbAPL Bioeng. 8, 046112 (2024); doi: 10.1063/5.0222866 8, 046112-5VC Author(s) 2024 02 December 2024 11:46:41pubs.aip.org/aip/apbfabrication” required for transplantation medicine. However, it isessential to introduce manipulation techniques such as micromanipu-lation because precise movements are required to assemble organoidunits.METHODSCell cultureAn easy-to-handle cancer cell line was used to validate the feasi-bility of fabricating the organoid units and their assembly. Green fluo-rescent protein (GFP)-labeled human breast adenocarcinoma cell line(MDA-MB-231; AKR-201, Cell Biolabs, San Diego, CA, USA) was cul-tured with Dulbecco’s modified eagle medium (DMEM; 31600-034,Gibco, Thermo Fisher Scientific, Waltham, MA, USA) containing 10%heat-inactivated fetal bovine serum (FBS; S1810, Biowest, Nuaill�e,France) and 1% penicillin-streptomycin (P/S; 15140-122, Gibco). Thiscell line has been used in our previous studies to form spheroids withcollagen substrates.25,44 Three types of human primary cells were alsoused to validate the construction of organ-like structures by co-culturing parenchymal and interstitial cells: human renal glomerularepithelial cells (HRGEpCs; 942-05n, Cell Applications, San Diego, CA,USA), human glomerular mesangial cells (NHMCs; ACBRI127, CellSystems, Kirkland, WA, USA), and human umbilical vein endothelialcells (HUVECs; 200-05n, Cell Applications). The primary cells werecultured with Medium 199 (31100-035, Gibco) containing 5% FBS, 1%P/S, 10lg/L human basic fibroblast growth factor (bFGF; GF-030-3,AUSTRAL Biologicals, San Ramon, CA, USA), 10lg/L human epider-mal growth factor (hEGF; E9644, Sigma-Aldrich, St. Louis, MO, USA),1% ITS-X supplement (094-06761, Fujifilm Wako Pure ChemicalCorp., Osaka, Japan), 36lg/L hydrocortisone (50-23-7, MPBiomedicals, Irvine, CA, USA), and 4ng/L 3,30,5-triiodo-L-thyroninesodium salt (T6397, Sigma-Aldrich). The cells were cultured in a75 cm2 flask (658175, Greiner Bio-One, Kremsm€unster, Austria) pre-coated with/without 0.1% bovine gelatin solution (G9391, Sigma-Aldrich) until reaching 90% confluence. Primary cells from the fifth toninth passages were used for experiments in this study.Mold processingMonolithic porous bulk material with superhydrophobicity(boehmite nanofiber-polymethylsilsesquioxane; BNF-PMSQ)44 wasprocessed using a CNC milling machine (monoFab SRM-20 or MDX-50, Roland DG, Shizuoka, Japan) to manufacture molds for variousshapes of organoids. We fabricated bead ring, cylinder, rectangularsolid, cubic, and sheet-shaped molds in addition to the sphericalone25,44 (supplementary Fig. 2). The mold models were created in a3D-CAD (SolidWorks, Dassault Syst�emes SOLIDWORKS Corp.,Waltham, MA, USA) and then exported in STL format, from whichthe processing paths were coded by CAM software (SRP Player,Roland DG). Processing was performed in two stages, roughing andfinishing, to prepare the surface of the monolithic porous material andmake it superhydrophobic (supplementary Table 1).Polytetrafluoroethylene (PTFE) was used as the mold material whennecessary.Organoid unit fabricationTo validate the feasibility of fabricating the organoid units andtheir assembly, the MDA-MB-231 cells or HRGEpCs were harvestedafter reaching 90% confluence with 0.25% trypsin-EDTA (25200-072,Gibco) or 0.05% trypsin-EDTA (25300-062, Gibco), respectively. Thecells were then resuspended in the culture medium at a concentrationof 5.0� 107 cells/ml. Cell-suspended collagen solution [4.0mg/ml;native collagen acidic solution (IAC-50, KOKEN, Tokyo, Japan), 10�DMEM, 10mM NaHCO3, 10mM HEPES-NaOH (pH7.5), and thecell suspension] was prepared on ice to give the final concentration of5.0� 106 cells/ml. The cell-suspended collagen solution was then dis-pensed onto the sterilized molds in a predetermined amount and order(Fig. 7). The dispensed solution was allowed to stand still in a CO2incubator (37 �C in a 100% humidified atmosphere of 5% CO2) for30–60min. After gelation, the primary organoid block was transferredto a 35mm diameter dish (3000-035, AGC Techno Glass, Shizuoka,Japan) by dropping a small amount of medium to cover it, poking itwith the tip end of a micropipette to float it, and then adding moremedium to pour it in. The units were incubated and matured for up to5 days while being observed under a stereomicroscope (SZX16,Olympus, Tokyo, Japan) or a fluorescence imaging system(THUNDER Imaging System, Leica Microsystems, Wetzlar,Germany).To validate the construction of organ-like structures with theunits in tri-culture, the HRGEpCs, NHMCs, and HUVECs were har-vested after reaching 90% confluence with 0.05% trypsin-EDTA andresuspended in the culture medium at a concentration of 5� 108 cells/ml. Cell-suspended protein solution [4.5mg/ml Matrigel (Matrix forOrganoid Culture, 356255, Corning, Corning, NY, USA), 1.5mg/mlnative collagen acidic solution, 10� DMEM, 10mM NaHCO3, 10mMHEPES-NaOH (pH7.5), and the cell suspensions] was prepared on iceto give the final concentrations of 0.5� 107 cells/ml (HRGEpCs andNHMCs) and 4.0� 107 cells/mL (HUVECs), respectively (i.e.,HRGEpCs:NHMCs:HUVECs¼ 1:1:8). Tri-cultured organoid units,similar to MDA-MB-231 units, were formed and gelatinized with theprepared solution. They were then transferred to 48-well plates (VTC-48, AS ONE Corp., Osaka, Japan) and cultured for 7 days usingOncoPro medium (A5701201, Gibco) supplemented with 1% P/S,10lg/L bFGF, and 10lg/L hEGF.Organoid unit assembly (unit construction)The units were first transferred to an empty tissue culture dishusing a micro spatula (6-524-01, AS ONE Corp.) or tweezers forblock-to-block assembly. We applied 5–10ll of adhesive to the area tobe bonded with a micropipette, stuck the units together, and incubatedfor 2–5min. Collagen solution [2.5mg/ml; native collagen acidic solu-tion, 10� DMEM, 10mM NaHCO3, 10mM HEPES-NaOH (pH7.5),and ultrapure water] was used as the adhesive for bonding MDA-MB-231 units, and protein solution [4.5mg/ml Matrigel, 1.5mg/ml nativecollagen acidic solution, 10� DMEM, 10mM NaHCO3, 10mMHEPES-NaOH (pH7.5), and the cell suspension (HUVECs; final con-centration, 1.0� 106 cells/ml)] was used for bonding tri-cultured units.After bonding, the assembled units were transferred to the 35mmdiameter dish filled with the culture medium using a medicine spoonto continue incubation.AntibodiesThe sheep polyclonal anti-nephrin antibody (Cat# AF4269) waspurchased from R&D Systems (Minneapolis, MN, USA). The mouseAPL Bioengineering ARTICLE pubs.aip.org/aip/apbAPL Bioeng. 8, 046112 (2024); doi: 10.1063/5.0222866 8, 046112-6VC Author(s) 2024 02 December 2024 11:46:41pubs.aip.org/aip/apbFIG. 3. Assembly of organoid units with various geometries. (a) Overview of bonding and stacking methods for organoid units. (b) Four-cylinder MDA-MB-231 organoid unitsbonded with collagen solution. Scale bar, 10 mm. (c) The jointed units can be pinched and lifted with tweezers without breaking into shreds. (d) Two rectangular solid MDA-MB-231 organoid units bonded together. Scale bar, 1 mm. (e) Three rectangular solid units were bonded and stacked and showed no signs of dissociation during the five-day cul-ture period. The stacking of the units was confirmed from the lateral view. Scale bar, 1 mm. (f) Live fluorescence images of cellular dynamics around the bonding area betweenthe units (upper row: overall view. Scale bar, 1 mm; lower row: magnified view. Scale bar, 500 lm). From day 1, cells that escaped from the units began to migrate into the colla-gen gel at the bonding area. Around day 4, cells proliferated at the bonding area, and the units connected in a cell cluster. The images were processed by the THUNDER imag-ing system with instant computational clearing (ICC) and extended depth of field (EDF). All data shown are representative of three independent experiments.APL Bioengineering ARTICLE pubs.aip.org/aip/apbAPL Bioeng. 8, 046112 (2024); doi: 10.1063/5.0222866 8, 046112-7VC Author(s) 2024 02 December 2024 11:46:41pubs.aip.org/aip/apbFIG. 4. Construction and assembly of human kidney glomerular tissue-like organoid units. (a) Fluorescence-stained frozen sections of tri-cultured spherical organoid units(n¼ 3). Each marker protein is stained on three cell types: NHMCs (CD90.1), HUVECs (CD31), and HRGEpCs (Nephrin). Scale bar, 50 lm. (b) Changes over culture durationin co-localization of marker proteins (mean 6 SD, n¼ 3; 8; or 9 images). (c) Overall view of fluorescence-stained frozen sections of tri-cultured spherical organoid units(n¼ 3). Scale bar, 500 lm. (d) Representative fluorescence-stained images of cellular dynamics around the bonding area between the tri-cultured cubic organoid units (n¼ 3,upper row: overall view. Scale bar, 1 mm; lower 5 rows: magnified view. Scale bar, 500 lm).APL Bioengineering ARTICLE pubs.aip.org/aip/apbAPL Bioeng. 8, 046112 (2024); doi: 10.1063/5.0222866 8, 046112-8VC Author(s) 2024 02 December 2024 11:46:41pubs.aip.org/aip/apbmonoclonal anti-CD31 antibody (Cat# 3528) was purchased from CellSignaling Technology (Danvers, MA, USA). The FITC-conjugatedhuman monoclonal anti-CD90.1 antibody (Cat# 130-112-683) waspurchased from Miltenyi Biotec (Bergisch Gladbach, Germany). AlexaFluor 594-conjugated goat anti-mouse IgG (Cat# A-11032) and AlexaFluor 647-conjugated donkey anti-sheep IgG (Cat# A-21448) second-ary antibodies were purchased from Thermo Fisher Scientific.The dilution concentrations of antibodies used in immunohisto-chemistry and immunofluorescence staining were as follows: nephrin,5lg/ml; CD31, 1:500; CD90.1, 1:30; secondary antibodies, 1:200. Theprimary antibodies against CD90.1, CD31, and nephrin label NHMCs,HUVECs, and HRGEpCs, respectively.ImmunohistochemistryCultured organoid units were fixed with 4% paraformaldehydephosphate buffer saline (PFA; 163-20145, Fujifilm Wako PureChemical Corp.) for 1 h at room temperature (RT). The units werecryoprotected by soaking in 20% sucrose/phosphate buffered saline(PBS; 05913, Nissui Pharmaceutical, Tokyo, Japan) for 5 h and 30%sucrose/PBS for an additional overnight at 4 �C. Fixed units were fro-zen in optical cutting temperature (OCT) compound (45833, SakuraFinetek Japan, Tokyo, Japan) and cut into 15lm-thick frozen sectionson cryofilm using a cryostat (CM1860, Leica Microsystems). After cut-ting out the frozen sections, the cells were permeabilized with 0.1%Triton X-100 (17-1315-01, Pharmacia Biotech, Uppsala, Sweden) inFIG. 5. Assembly of organoid units with different geometries. (a) Overview of how to assemble the organoid units with different geometries. (b) Time-sequenced brightfieldimages of the assembled MDA-MB-231 organoid units (n¼ 3). Scale bar, 1 mm. (c) Live fluorescence images of the assembled MDA-MB-231 organoid units, expressing GFPin the cytoplasm (n¼ 3). Upper row: wide-field fluorescence images (raw data), lower row: images processed by the THUNDER imaging system with instant computationalclearing (ICC) and extended depth of field (EDF). Scale bars, 1 mm. (d) Representative fluorescence-stained images of the assembled human-kidney glomerular tissue-likeorganoid units (n¼ 3). Each marker protein is stained on three cell types: NHMCs (CD90.1), HUVECs (CD31), and HRGEpCs (Nephrin). Upper 5 rows: wide-field fluorescenceimages (raw data), lower row: images processed by the THUNDER imaging system with instant computational clearing (ICC) and extended depth of field (EDF). Scale bar,1 mm.APL Bioengineering ARTICLE pubs.aip.org/aip/apbAPL Bioeng. 8, 046112 (2024); doi: 10.1063/5.0222866 8, 046112-9VC Author(s) 2024 02 December 2024 11:46:41pubs.aip.org/aip/apbPBS, followed by incubation in 1% Block Ace (BA; UKB40, KAC,Kyoto, Japan) in PBS to prevent nonspecific antibody absorption. Thecells were then stained using the primary and secondary antibodiesdiluted in 1% BA in PBS and PBS, respectively. Cell nuclei were stainedusing 40,6-diamidino-2-phenylindole (DAPI; D1306, Invitrogen,Thermo Fisher Scientific). Stained organoid unit sections wereobserved with optical sectioning fluorescence microscopy (AxioObserver 7 with Apotome 3, Carl Zeiss, Oberkochen, Germany).FIG. 6. Concept of the proposed unit construction method for replication of full-scale organs with organoids.FIG. 7. Procedure for fabrication of organoid units with each geometry. Cell-suspended collagen/protein solution was prepared on ice to give the final concentration of5.0� 106 cells/ml in this study.APL Bioengineering ARTICLE pubs.aip.org/aip/apbAPL Bioeng. 8, 046112 (2024); doi: 10.1063/5.0222866 8, 046112-10VC Author(s) 2024 02 December 2024 11:46:41pubs.aip.org/aip/apbImmunofluorescence stainingCultured organoid units were fixed with 4% PFA for 1 h. Formultiplex staining with antibodies, the samples were made transparent.After fixation, the units were immersed overnight in 50% Tissue-Clearing Regent CUBIC-L (T3740, Tokyo Chemical Industry, Tokyo,Japan) containing 500mM NaCl, followed by membrane permeabili-zation and blocking with 0.15% Triton-X100 and 1% BA in PBS for 1h. The cells were stained overnight at 4 �C with primary antibodies(diluted in 1% BA in PBS) and secondary antibodies (diluted in PBS),respectively, and then post-fixed with 4% PFA for 1 h. Cell nuclei werealso stained with DAPI. The stained units were soaked in 50% Tissue-Cleaning Reagent CUBIC-Rþ(N) (T3983, Tokyo Chemical Industry)for 20min and observed in 100% CUBIC-Rþ(N). Unless otherwisenoted, the staining process was performed at RT, with three 15-minwashes with PBS between each step. All processes were carried outwith shaking. Fluorescence images of the stained units were obtainedwith the fluorescence imaging system or optical sectioning fluores-cence microscopy.Live/dead assayThe cell death in the organoid units was quantitatively evaluatedusing an Apoptotic, Necrotic, and Healthy Cells Quantification Kit(30018, Biotium, CA, USA) according to the manufacturer’s instruc-tions. The 2mm-diameter spherical organoid units prepared withMDA-MB-231 or HRGEpC were washed with PBS with Mg2þ andCa2þ thrice, followed by incubation with the staining solution contain-ing FITC-Annexin V, Ethidium homodimer III, and Hoechst 33342for 30min at 37 �C. The cells were then washed with the PBS withMg2þ and Ca2þ thrice, and fixed with PFA containing 1.25mM CaCl2for an hour. The stained cells were covered with the PBS with Mg2þand Ca2þ after washing out PFA.We observed their fluorescence usingthe fluorescence imaging system. MDA-MB-231 cells are a stableGFP-expressing cell line and, therefore, could not be labeled withFITC-Annexin V and were only stained with Hoechst and Ethidiumhomodimer III.Data quantificationTo measure the overall changes, i.e., morphological changes, inorganoid units, we monitored their area, perimeter, height, and majorand minor axes based on stereomicroscopic images using ImageJFiji.45 In the cases of bead ring, sphere, and spherical shell units, theirdiameters (i.e., major and minor axes) were obtained by a computationbased on an ellipse equivalent to the outline shape. For the bead shapeunits, the major and minor axes of the outer and inner circumferenceswere measured, respectively. For the spherical shell units, the majorand minor axes of the outer and inner spheres were measured,respectively.To evaluate the spatial relationship among different cell typeswithin the organoid unit, which contains a large number of cells, weused the standard index of co-localization, the Pearson’s correlationcoefficient.46,47 The correlation coefficient was obtained from the opti-cal sectioning fluorescence microscopy data using ImageJ Fiji’s “Coloc2” function.Data reproducibilityAll values are shown as mean 6 standard deviation (SD) unlessstated otherwise. Data were obtained from at least three independentlyrepeated experiments.SUPPLEMENTARY MATERIALSee the supplementary material for the details of the molds usedto fabricate the organoid units, the conditions for milling them, andthe descriptions of supplementary movies.ACKNOWLEDGMENTSThis study was partly supported by grants from the JSPSKAKENHI (Grant No. 21K19893 to D.Y.), the JST FORESTProgram (Grant No. JPMJFR222S to D.Y.), and NIMS JointResearch Hub Program (Grant No. 2023-097 to G.H.). The authorswould like to thank Science Graphics Co., Ltd. for preparing andediting part of the figures (Fig. 6).AUTHOR DECLARATIONSConflict of InterestThe authors have no conflicts to disclose.Ethics ApprovalEthics approval is not required.Author ContributionsAyaka Kadotani: Conceptualization (equal); Investigation (lead);Methodology (lead); Validation (lead); Visualization (lead); Writing –original draft (equal); Writing – review & editing (equal). Gen Hayase:Funding acquisition (supporting); Resources (supporting); Supervision(supporting); Writing – review & editing (equal). Daisuke Yoshino:Conceptualization (equal); Funding acquisition (lead); Methodology(supporting); Project administration (lead); Resources (lead);Supervision (lead); Validation (supporting); Visualization (support-ing); Writing – original draft (equal); Writing – review & editing(equal).DATA AVAILABILITYThe data that support the findings of this study are availablewithin the article and its supplementary material.REFERENCES1J. Kim, B. K. Koo, and J. A. Knoblich, “Human organoids: Model systems forhuman biology and medicine,” Nat. Rev. Mol. Cell Biol. 21, 571–584 (2020).2M. Takasato, P. Er, H. Chiu et al., “Kidney organoids from human iPS cellscontain multiple lineages and model human nephrogenesis,” Nature 526, 564–568 (2015).3M. Lancaster, M. Renner, C. A. 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